1. Field of the Invention
The presently claimed and disclosed invention relates, in general, to dual action heparin synthases and, more particularly, to dual action heparin synthases obtained from Pasteurella multocida. The presently claimed and disclosed invention also relates to heparosan, heparin and heparin-like molecules produced according to recombinant techniques and methods of using such molecules. The presently claimed and disclosed invention also relates to methods, and molecules produced according to such methods, for using the presently claimed and disclosed heparosan and/or heparin synthases for polymer grafting and the production of non-naturally occurring chimeric polymers incorporating stretches of one or more acidic GAG molecules, such as heparin, chondroitin, hyaluronan, and/or heparosan.
2. Background Information Relating to this Application
Glycosaminoglycans [GAGs] are long linear polysaccharides consisting of disaccharide repeats that contain an amino sugar and are found in most animals. Chondroitin [β(1,4)GlcUA-β(1,3)GalNAc]n, heparin/heparosan [β1,4)GlcUA-[α(1,4)GlcNAc]n, and hyaluronan [β(1,4)GlcUA-β(1,3)GlcNAc]n are the three most prevalent GAGs found in humans and are also the only known acidic GAGs. Chondroitin and heparin typically have n=20 to 100, while hyaluronan typically has n=103. Chondroitin and heparin are synthesized as glycoproteins and are sulfated at various positions in vertebrates. Hyaluronan is not sulfated in vertebrates. A substantial fraction of the GlcUA residues of heparin and chondroitin are epimerized to form iduronic acid. A simplified nomenclature has been developed for these GAGs. For example, heparin/heparosan's structure is noted as β4-GlcUA-α4-GlcNAc.
The capsular polysaccharide produced by the Type D strain of Pasteurella multocida is N-acetyl heparosan (heparosan is unmodified heparin—i.e. sulfation or epimerization have not occurred). In vertebrates, one or more modifications including O-sulfation of certain hydroxyls, deacetylation and subsequent N-sulfation, or epimerization of glucuronic acid to iduronic acid modifies the precursor N-acetyl heparosan to heparin/heparan. Hereinafter, for convenience and/or ease of discussion, heparin and/or heparosan are defined as polymers having the β4GlcUA-α4GlcNAc backbone.
Many lower animals possess these same GAGs or very similar molecules. GAGs play both structural and recognition roles on the cell surface and in the extracellular matrix. By virtue of their physical characteristics, namely a high negative charge density and a multitude of polar hydroxyl groups, GAGs help hydrate and expand tissues. Numerous proteins bind selectively to one or more of the GAGs. Thus the proteins found on cell surfaces or the associated extracellular matrices (e.g. CD44, collagen, fibronectin) of different cell types may adhere or interact via a GAG intermediate. Also GAGs may sequester or bind certain proteins (e.g. growth or coagulation factors) to cell surfaces.
Certain pathogenic bacteria produce an extracellular polysaccharide coating, called a capsule, which serves as a virulence factor. In a few cases, the capsule is composed of GAG or GAG-like polymers. As the microbial polysaccharide is identical or very similar to the host GAG, the antibody response is either very limited or non-existent. The capsule is thought to assist in the evasion of host defenses such as phagocytosis and complement. Examples of this clever strategy of molecular camouflage are the production of an authentic HA polysaccharide by Gram-negative Type A Pasteurella multocida and Gram-positive Group A and C Streptococcus. The HA capsule of these microbes increases virulence by 102 to 103-fold as measured by LD50 values, the number of colony forming units that will kill 50% of the test animals after bacterial challenge.
The invasiveness and pathogenicity of certain E. coli strains has also been attributed to their polysaccharide capsules. Two Escherichia coli capsular types, K4 and K5, make polymers composed of GAG-like polymers. The E. coli K4 polymer is an unsulfated chondroitin backbone decorated with fructose side-branches on the C3 position of the GlcUA residues. The K5 capsular material is a polysaccharide, called heparosan, identical to mammalian heparin except that the bacterial polymer is unsulfated and there is no epimerization of GlcUA to iduronic acid.
The studies of GAG biosynthesis have been instrumental in understanding polysaccharide production in general. The HA synthases were the first GAG glycosyltransferases to be identified at the molecular level. These enzymes utilize UDP-sugar nucleotide substrates to produce large polymers containing thousands of disaccharide repeats. The genes encoding bacterial, vertebrate, and viral HAS enzymes have been cloned. In all these cases, expression studies have demonstrated that transformation with DNA encoding a single HAS polypeptide conferred the ability of foreign hosts to synthesize HA. Except for the most recent HAS to be identified, P. multocida pmHAS, these proteins have similar amino acid sequences, repeating conserved amino acid motifs, and predicted topology in the membrane. Likewise, as presently disclosed and claimed herein, heparosan and/or heparin synthases have been identified that confer upon a foreign host the ability to produce heparin.
With respect to related microbial GAG synthases other than the HASs, the E. coil K5 heparin glycosyltransferases, KfiA (SEQ ID NO:8) and KfiC (SEQ ID NO:9), have been identified by genetic and biochemical means. These K5 glycosyltransferases synthesize heparosan (unsulfated and unepimerased heparin) in vivo. The KfiA and KfiC require KfiB (SEQ ID NO:10), an accessory protein, with unknown function in order to synthesize heparosan, however. In vitro, the reactions are limited to adding one or two sugars; as such, it appears that some co-factor or reaction condition is missing—thus, extended polymerization does not occur in vitro when KfiA, KfiB, and KfiC are used. As such, the presently claimed and disclosed heparosan/heparin synthases provide a novel heretofore unavailable means for recombinantly producing heparin (the sulfated and epimerized molecule). In contrast to the HASs, the pmCS chondroitin synthase(s), and the presently disclosed and claimed heparin synthases, it appears that K5 requires two proteins, KfiA and KfiC, to transfer the sugars of the disaccharide repeat to the growing polymer chain. The presently claimed and disclosed heparin synthases (designated “pmHS and PglA”) are dual action enzymes capable of transferring both sugars of the growing heparin polymer chain. These enzymes polymerize heparosan in vivo and in vitro.
Many P. multocida isolates produce GAG or GAG-like molecules as assessed by enzymatic degradation and removal of the capsule of living bacterial cells. Type A P. multocida, the major fowl cholera pathogen, makes a capsule that is sensitive to hyaluronidase. Subsequent NMR structural studies of capsular extracts confirmed that HA was the major polysaccharide present. A specific HA-binding protein, aggrecan, also interacts with HA from Type A P. multocida. Two other distinct P. multocida types, a swine pathogen, Type D, and a minor fowl cholera pathogen, Type F, produce polymers that are chondroitin or chondroitin-like based on the observation that their capsules are degraded by Flavobacterium chondroitin AC lyase. After enzymatic removal of the capsule, both types were more readily phagocytosed by neutrophils in vitro. The capsule of Type D cells, but not Type F cells, also appear to be degraded by heparinase III, indicating that a heparin-type molecule is present as well.
Heparin acts as an anticoagulant and is used to avoid coagulation problems during extra corporal circulation and surgery as well as for treatment after thrombosis has been diagnosed. Heparin is used in the prevention and/or treatment of deep venous thrombosis, pulmonary embolism, mural thrombus after myocardial infarction, post thrombolytic coronary rethrombosis, unstable angina, and acute myocardial infarction. In addition to use as a treatment for various medical conditions, heparin is also used to coat medical instruments and implants, such as stents, to prevent blood clotting. Using heparin to coat various medical items eliminates the need to prescribe anti-clotting medication in some cases.
Where heparin is used to treat medical conditions as those described above, two different methods and two different types of heparin are used. The two methods are intravenous infusion of standard heparin and injection of low molecular mass heparin. Patients undergoing intravenous infusion are hospitalized and the activated partial thromoplastin time (aPTT) is monitored. This type of treatment requires that the patient remain hospitalized until warfarin is administered to achieve an International Normalized Ratio (INR) between 2.0 and 3.0 often resulting in a three to seven day hospital stay. The alternative treatment involves twice daily injections of low-molecular-weight heparin. The injection treatment allows the patient to self-administer or have a visiting nurse or family member administer the injections.
Low molecular weight heparin has a molecular weight of 1,000 to 10,000 Daltons as compared to the molecular weight of standard heparin of 5,000 to 30,000 Daltons. Low molecular weight heparin binds less strongly to protein than standard heparin, has enhanced bioavailability, interacts less with platelets and yields more predictable blood levels. The predictability of blood levels eliminates the need to monitor the aPPT. In addition, low molecular weight heparin offers a lower likelihood of bleeding and no reports of thrombocytopenia or osteoporosis have been issued with respect to low molecular weight heparin.
In the presently claimed and disclosed invention, the monosaccharide composition of the P. multocida Type D polysaccharide has been identified and analyzed. The DNA sequence information of the Type A HA biosynthesis locus and the Type F biosynthesis locus allowed for the prediction of the general properties of the Type D locus. From this information on potential precursor genes required by a heparin synthase, pmHS was identified (P. multocida Heparin Synthase), the first dual action microbial heparin synthase to be identified and molecularly cloned from any source. With respect to the pmHS, a single polypeptide is responsible for the copolymerization of the GlcUA and GlcNAc sugars—i.e. it is a dual action enzyme as opposed to the single action nature of the at least three enzymes of E. coli K5 heparosan biosynthesis locus that are required for heparin production. The identification of pmHS also allowed for the identification of the existence of another heparin synthase found in Type A, D and F P. multocida. A gene with unknown function, called PglA, was found in a genome sequencing project of Type A P. multocida; no enzymatic function (or any function) has been previously described with respect to this PglA gene. Hereinafter, and is contemplated and included within the presently disclosed and claimed invention, is disclosed that the PglA enzyme, which is 70% identical to pmHS, is also a heparin synthase. This unexpected cryptic gene is functional in vitro in recombinant systems. The Type D capsular polymer has been identified as a heparin polymer. Organisms with the heparin synthase gene (Type D P. multocida) as new sources of heparin polymer have also been identified, purified, and characterized.